Nanoendoscopy-AFM probes nuclear elasticity in living lung cancer cells

Researchers at Kanazawa University have used nanoendoscopy atomic force microscopy to create nanoscale maps of nuclear stiffness in living lung cancer cells

A research team at the Nano Life Science Institute (NanoLSI), Kanazawa University, Kanazawa, Japan, has reported a high precision, novel method to measure nuclear elasticity – the stiffness or softness of the cell nucleus – in living cells. They used nanoendoscopy atomic force microscopy (NE-AFM) – a variant of atomic force microscopy that inserts a nanoneedle probe directly into cells – to reveal how cancer cell nuclei stiffen, or soften, according to chromatin structure and environmental conditions.

The findings provide fundamental insight into how the physical properties of cancer cell nuclei change during disease progression and indicate that nuclear mechanics could serve as a measurable biomarker for diagnosis and for the evaluation of treatment response. Changes in nuclear mechanics are a recognised hallmark of cancer and can indicate malignant transformation well before gross changes in tissue architecture become apparent.

Until now, most studies of nuclear elasticity have relied on atomic force microscopy (AFM) probes that press on the outer cell membrane, or on aspiration experiments that deform isolated nuclei. Both approaches have their limitations. AFM measurements at the cell surface are strongly influenced by the cytoskeleton and other structures around the nucleus, while experiments on isolated nuclei cannot fully reproduce the intact physical state of the nucleus inside a living cell.

The NE-AFM method developed by Takehiko Ichikawa and colleagues at Kanazawa University has addressed these constraints. The technique inserts an ultrafine nanoneedle probe directly into living cells thousands of times without severe damage, so the team could map nuclear elasticity at the nanoscale under conditions that closely resemble the in vivo environment.

In their study, the researchers examined human lung cancer cells of the PC9 line. They found that PC9 cell nuclei became markedly stiffer under serum-free culture conditions. This increase in nuclear elasticity correlated with higher levels of trimethylation of histone H4 at lysine 20 (histone H4K20me3), a chemical modification associated with compact, transcriptionally inactive chromatin.

When the team exposed cells to transforming growth factor beta (TGF-β), which induces epithelial–mesenchymal transition (EMT) and promotes a more migratory, invasive cellular phenotype, the nuclei became softer and histone H4K20me3 levels fell. The work therefore showed that changes in nuclear elasticity arise primarily from shifts in chromatin compaction states rather than from alterations in the nuclear lamina proteins that line the inner nuclear membrane.

The group then evaluated brain-metastatic derivatives of PC9 cells, termed PC9-BrM. These metastatic cells showed similar trends in nuclear elasticity and histone modification status which suggests that chromatin-based control of nuclear mechanics contributes to invasive behaviour and metastatic potential. The link between a biochemical marker of chromatin compaction and a mechanical property of the nucleus provides a bridge between epigenetic regulation and the physical forces that act within cancer cells as they invade and spread.

To implement nanoendoscopy-AFM, the researchers combined conventional AFM hardware with specialised nanoneedle probes fabricated by electron beam deposition. Each probe had a diameter of about 160 nanometres, narrow enough to penetrate the cell and nucleus with minimal disruption. The probes entered living cells repeatedly to record thousands of force–distance curves across the nuclear surface. From these data, the team reconstructed three-dimensional maps of nuclear stiffness in intact human lung cancer cells. Unlike traditional AFM approaches at the cell surface, NE-AFM can distinguish cell membrane elasticity from nuclear elasticity and avoids interference from cytoskeletal elements beneath the plasma membrane.

The mechanical measurements were supported by biochemical analyses. The researchers used immunoblotting to correlate local changes in elasticity with specific histone modifications and with nuclear protein levels, including the lamins that form the structural scaffold of the nuclear envelope.

This combined approach strengthened the conclusion that chromatin compaction, rather than changes in the lamina, plays the dominant role in the observed shifts in nuclear mechanics under different culture conditions and during EMT.

“Our work shows that nuclear elasticity is not just a physical property but a reflection of underlying chromatin states.

“With Nanoendoscopy-AFM, we now have a powerful tool to directly probe the nucleus of living cancer cells. This opens the door to novel diagnostic approaches and to a better understanding of how mechanical forces shape cancer progression,” said Ichikawa.

The research has demonstrated that nuclear elasticity can act as a practical biomarker of cancer progression, with potential relevance for early diagnosis and prognosis. In principle, clinicians could one day use similar nanoscale mechanical measurements to stratify tumours according to their metastatic potential or to monitor the response of cancer cells to chemotherapy, targeted agents or immunotherapy. Beyond oncology, the NE-AFM platform offers an opportunity to study chromatin regulation and nuclear mechanics in developmental biology, immunology and neurodegeneration, where nuclear structure and chromatin state also have central roles.

The ability to insert a nanoneedle into living cells with high precision also suggests further applications. Nanoendoscopy-AFM could help researchers to explore the mechanical properties of other organelles – such as mitochondria – or to investigate how local forces within the cytoplasm influence signal transduction and gene expression. As NE-AFM is compatible with live-cell imaging and standard cell culture conditions, it may integrate with fluorescence microscopy or high-content screening to provide both mechanical and molecular readouts in parallel.

The study rests on several key concepts in cell and cancer biology. A scanning nanoprobe microscope, such as the NE-AFM platform, uses a nanometre-scale probe to interrogate structures inside living cells with very high spatial precision. Nuclear elasticity refers to how stiff or soft a cell nucleus is under an applied force; it reflects chromatin organisation and the architecture of the nuclear lamina and can vary markedly between healthy and malignant cells.

Chromatin compaction describes how DNA and its associated histone proteins are packed within the nucleus; tightly packed chromatin tends to silence genes and stiffen the nucleus, while more open chromatin often permits active transcription and yields a softer nucleus. Histone H4K20me3 is a specific chemical modification of histone H4 that typically marks compact, transcriptionally inactive chromatin and – in this study – correlated with stiffer nuclei in lung cancer cells.

For further reading please visit: 10.1021/acsanm.5c03044